Gene expression encompasses a number of steps originating from the DNA template, ultimately to the final protein or protein product. Control and regulation of gene expression can occur through numerous mechanisms. The initiation of transcription of a gene is generally thought of as the predominant control of gene expression. Transcriptional controls (or promoters) are generally short sequences embedded in the 5′-flanking or upstream region of a transcribed gene. There are promoter sequences which affect gene expression in response to environmental stimuli, nutrient availability, or adverse conditions including heat shock, anaerobiosis or the presence of heavy metals. There are also DNA sequences which control gene expression during development, or in a tissue, or in an organ specific fashion, and, of course there are constitutive promoters.
Promoters contain the signals for RNA polymerase to begin transcription so that protein synthesis can proceed. DNA binding, nuclear, localized proteins interact specifically with these cognate promoter DNA sequences to promote the formation of the transcriptional complex and eventually initiate the gene expression process. The entire region containing all the ancillary elements affecting regulation or absolute levels of transcription may be comprised of less than 100 base pairs or as much as 1 kilobase pairs.
One of the most common sequence motifs present in the promoters of genes is the “TATA” element which resides upstream of the start of transcription. Promoters are also typically comprised of components which include a TATA box consensus sequence at about 35 base pairs 5′ relative to the transcription start site or cap site which is defined as +1. The TATA motif is the site where the TATA-binding-protein (TBP) as part of a complex of several polypeptides (TFIID complex) binds and productively interacts (directly or indirectly) with factors bound to other sequence elements of the promoter. This TFIID complex in turn recruits the RNA polymerase II complex to be positioned for the start of transcription generally 25 to 30 base pairs downstream of the TATA element and promotes elongation thus producing RNA molecules.
In most instances sequence elements other than the TATA motif are required for accurate transcription. Such elements are often located upstream of the TATA motif and a subset may have homology to the consensus sequence CCAAT.
Promoters are usually positioned 5′ or upstream relative to the start of the coding region of the corresponding gene, and the entire region containing all the ancillary elements affecting regulation or absolute levels of transcription may be comprised of less than 100 base pairs or as much as 1 kilobase pair.
A number of promoters which are active in plant cells have been described in the literature. These include nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor inducing plasmids of Agrobacterium tumefaciens) The cauliflower mosaic virus (CaMV) 19S and 35S promoters, the light-inducible promoter from the small subunit of ribulose bisphosphate carboxylase (ssRUBICSO, a very abundant plant polypeptide), the alcohol dehydrogenase (AdhI and AdhII) promoters from maize, and the sucrose synthase promoter. All of these promoters have been used to create various types of DNA constructs which have been expressed in plants. (See for example PCT publication WO84/02913 Rogers, et al). Perhaps the most commonly used promoter is the 35S promoter of Cauliflower Mosaic Virus. The (CaMV) 35S promoter is a dicot virus promoter, however, it directs expression of genes introduced into protoplasts of both dicots and monocots. The 35S promoter is a very strong promoter and this accounts for its widespread use for high level expression of traits in transgenic plants. The CaMV35S promoter however has also demonstrated relatively low activity in several agriculturally significant graminaceous plants such as wheat.
The promoters of the maize genes encoding alcohol dehydrogenase, AdhI and AdhII, have also been widely used in plant cell transformations. Both genes are induced after the onset of anaerobiosis. Maize AdhI has been cloned and sequenced as has been AdhII. Formation of an AdhI chimeric gene, Adh-CAT comprising the AdhI promoter linked to the chloramphenicol acetyltransferase (CAT) coding sequences and nopaline synthase (NOS) 3′ signal caused CAT expression at approximately 4-fold higher levels at low oxygen concentrations than under control conditions. Sequence elements necessary for anaerobic induction of the ADH-CAT chimeric have also been identified. The existence of anaerobic regulatory element (ARE) between positions −140 and −99 of the maize AdhI promoter composed of at least two sequence elements at positions −133 to −124 and positions −113 to −99 both of which have found to be necessary and are sufficient for low oxygen expression of ADH-CAT gene activity. The Adh promoter however responds to anaerobiosis and is not a constitutive promoter, drastically limiting its effectiveness.
Yet another important promoter in plants is the maize ubiquitin promoter which is described in U.S. Pat. No. 5,510,474, to Quail et al. the disclosure of which is incorporated herein by reference (SEQ ID NO:15). This promoter has become widely used in transgenic plant protocols. The promoter, as described in the patent, comprises RNA polymerase recognition and binding sites, a transcriptional initiation sequence (cap site), regulatory sequences responsible for inducible-transcription, an untranslatable intervening sequence (intron) between the transcriptional start site and the translational initiation site, and two overlapping heat shock consensus promoter sequences 5′ (−214 and −204) of the transcriptional start site. The entire promoter is almost 2 kb in length and has been shown to be functional in both monocot and dicot plants. The sequence of the maize ubiquitin promoter is disclosed in Quail et al. Expression levels achieved with the ubiquitin (Ubi-1) promoter driving the CAT gene in oat protoplast cells were higher than those of the CaMV promoter (Quail et al.).
There is a continuing need in the art for high level expression promoters, as well as promoters which are spatially defined in their expression patterns.
Expression of foreign nucleotide sequences introduced to cells must achieve more than a basal expression rate to produce enough protein to effect the desired phenotype or to harvest from the cell.
It is a primary object of this invention to provide novel maize Ubi-1 promoter sequences that increase expression of introduced genes in plant cells and plant tissues, compared to the non-engineered promoter.
It is yet another object of the invention to provide promoter sequences which result in expression in transgenic plants which unexpectedly alters or reverses the ratio of endosperm/embryo expression from known Ubi-1 promoters in the seed of regenerated plants.
It is an object of this invention to provide recombinant promoter molecules that provide for reliably high levels of expression of introduced genes in target cells.
It is yet another object of this invention to provide plants, plant cells and plant tissues containing the recombinant promoter of the invention.
It is yet another object of the invention to provide vehicles for transformation of plant cells including viral or plasmid vectors and expression cassettes incorporating the novel promoter sequences of the invention.
It is yet another object of the invention to provide bacterial cells comprising such vectors for maintenance, replication, and plant transformation.
Other objects of the invention will become apparent from the description of the invention which follows.